Toxoplasma gondii and related Apicomplexan parasites including Cryptosporidium cause significant human disease particularly in AIDS patients. T. gondii and C. parvum are also category B bioterrorism agents that pose a particular threat to the immunocompromised. The Apicomplexa are named for their unique apical secretory organelles-micronemes, rhoptries and dense granules. Proteolytic processing of secretory organelle contents occurs throughout the Apicomplexa. We have characterized two subtilisin-like serine proteinases (subtilases) from T. gondii, TgSUB1 and TgSUB2. TgSUB1 localizes to the micronemes, whereas TgSUB2 is a rhoptry protein that appears to be a rhoptry protein maturase. TgSUB2 cannot be disrupted, and antisense depletion of TgSUB2 results in parasites that divide poorly, have few rhoptries, and have large aberrant cytoplasmic vesicles. In contrast, TgSUB1 disruptants are viable. We hypothesize that Toxoplasma subtilases play an essential role in the biogenesis or function of micronemes and rhoptries. We plan to test the importance of TgSUB2 using genetic methods and to characterize TgSUB2 biological substrates. Because proteinase inhibitors typically mimic substrates, identification of a natural substrate for TgSUB2 would accelerate synthesis of specific inhibitors and elucidate the role of TgSUB2 in parasite survival. We will use recombinant TgSUB2 to develop an in vitro assay for TgSUB2 activity that can be used to identify substrates and unique inhibitors of TgSUB2. Finally, we will investigate the expression of other subtilase genes present in the T. gondii genome. We hypothesize that TgSUB1 is not essential because its function is redundant with other subtilase family members. We will determine the expression pattern and localization of other subtilases in T. gondii tachyzoites and use genetic techniques to determine if they have overlapping function with TgSUB1. In addition to illuminating the function and biology of T. gondii secretory organelles, these studies will determine whether subtilases are a viable chemotherapeutic target for treatment of human infections with Apicomplexan parasites.